Chemical Arsenal Research Articles

The Potential Role of Reactive Oxygen Species Produced by Low-Density Neutrophils in Periodontitis.

Neutrophils own an arsenal of dischargeable chemicals that enable them to handle bacterial challenges, manipulating innate immune response and actual participation in acquired immunity. The reactive oxygen species (ROS) are one of the most important chemicals that neutrophils discharge to eradicate pathogens. Despite their beneficial role, the ROS were strongly correlated to periodontal tissue destruction. Lowdensity neutrophils (LDN) have been recognized for producing enhanced quantities of ROS. However, the potential role of ROS produced by LDN in periodontitis is unknown. The aim of the study was to investigate the impact of ROS produced by LDN in periodontal diseases. Venous blood and periodontal parameters were obtained from 100 systemically healthy subjects divided into 40 participants with healthy periodontium in the control group and 60 with unstable periodontitis in the study group. Flow cytometry was used to measure the production of ROS by LDN in both groups. The data were analyzed for normal distribution using the Shapiro-Wilk test at p < 0.05, Spearman's correlations, and Mann-Whitney U test. Statistical analysis was performed in SPSS v25. No difference between the groups had been obtained in ROS production by LDN. However, a significant positive correlation existed between ROS and clinical attachment loss in periodontitis. LDN exhibits the same ROS generation capacity in the control and periodontitis groups.

Metal-Free Heterogeneous Photocatalysis for Carbocarboxylation of Alkenes: Efficient Synthesis of γ-Amino Carboxylic Derivatives.

A metal-free heterogeneous protocol is established herein for the synthesis of value-added γ-amino acid scaffolds via carbocarboxylation of alkenes with CO2 and alkylamines under visible light irradiation. The protocol shows broad substrate scope under mild reaction conditions and good stability of the catalyst for recycle tests. Moreover, the methodology could be feasible to the late-stage derivatization of several natural products, enriching the chemical arsenal for practical application.

An In Silico Comparative Study of Eleven Wasp Venom Allergens: An Arena for Therapeutic Approach

Peptide toxins in animals as part of the chemical arsenal for predation and/or protection, and they can also safeguard the host from pathogenic infections. Insects including the hymenopterans generate a battery of toxic bactericidal or bacteriostatic molecules which are small multifunctional, linear peptides that cause pain, have antimicrobial effects, and inflammatory processes . The toxins under the experiment are active against gram-positive, gram-negative bacteria and fungi. The present in silico study aims to predict the physicochemical attributes like molecular weight, theoretical pI, amino acid composition, extinction co-efficient, estimated half-life, instability index, aliphatic index and grand average of hydropathy (gravy) of wasps’ (class: Insecta; order: Hymenoptera) 11 wasp venom allergens through Expasy Protparam &amp; Pepstat software tools. The secondary structures of the toxins were predicted using psi-Blast-based secondary structure prediction GOR4 tools revealing the % α helix, extended β strand, random coil and ambiguous state reflecting a comparative picture of physicochemical parameters of these defensins. 3D Homology modelling of these toxins was accomplished through Swiss-model webserver tool and validated through various in silico tools like ANOLEA, ProSA-web, QMean4 determining Z score, PROCHEK establishing the 3D models of these toxins. Use of Inter Pro, CDD, PROSITE, P fam, Tox DL, PrDOS software predicted the protein family, protein toxicity, protein disorder respectively. Scratch Protein Predictor software tools predicted cysteine – cysteine bonds. Docking of the eleven (11) wasp venom allergens individually with bacterial cell wall component N-acetylglucosamine was done by CB DOCK webserver resulting negative affinity scores reflecting towards the strong binding between the mentioned 11 toxins and N-acetylglucosamine indicating that the mentioned wasps’ toxin molecules might be used as potential antibacterial therapeutic molecules binding to N-acetylglucosamine leading to an avenue to the probable bacterial drug discovery.

Application Of Wasps’ Defensins As Anti-Fungal Therapeutic Molecules: An In Silico Comparative Study

Animal peptide toxins as part of chemical arsenal for predation and/or protection that can safeguard host from pathogenic infections. Hymenopterans generate toxic bactericidal or bacteriostatic molecules, called defensins are small multifunctional, linear, polycationic peptides causing pain, have antimicrobial effects. Defensins are active against gram-positive, gram-negative bacteria and fungi. The present in silico study aims to predict the physicochemical attributes like molecular weight, theoretical pI, amino acid composition, extinction co-efficient, estimated half-life, instability index, aliphatic index and grand average of hydropathy (gravy) of 09 different wasps’ defensins using Expasy Protparam tool. The secondary structures of the toxins were predicted using psi-Blast-based PSIPRED, SOPMA tools revealing % α helix, extended β strand, random coil and ambiguous state reflecting a comparative physico-chemical parameters of these defensins. 3D Homology modelling of these toxins was accomplished through Swiss-model webserver and validated through ProSA-web, QMean4 determining Z score,PROCHEK establishing the 3D models of these defensins. Use of InterPro, CDD, ToxDL, PrDOS software predicted protein family, conserved domain, protein toxicity, protein disorder respectively. CYSCON and CYSPRED tools predicted cysteine-cysteine bonds. Docking of the nine (09) wasps’ defensins individually with fungal cellwall component 1,4 Beta-D-Glucan was done by DockThor webserver resulting negative affinity scores refleting strong binding between the defensins and 1,4 beta-D-Glucan indicating that the mentioned wasps’ defensin molecules might be used as potential antifungal therapeutic molecules binding to 1, 4 Beta-D-Glucan indicating an avenue to antifungal drug discovery.

Venom biotechnology: casting light on nature's deadliest weapons using synthetic biology.

Venoms are complex chemical arsenals that have evolved independently many times in the animal kingdom. Venoms have attracted the interest of researchers because they are an important innovation that has contributed greatly to the evolutionary success of many animals, and their medical relevance offers significant potential for drug discovery. During the last decade, venom research has been revolutionized by the application of systems biology, giving rise to a novel field known as venomics. More recently, biotechnology has also made an increasing impact in this field. Its methods provide the means to disentangle and study venom systems across all levels of biological organization and, given their tremendous impact on the life sciences, these pivotal tools greatly facilitate the coherent understanding of venom system organization, development, biochemistry, and therapeutic activity. Even so, we lack a comprehensive overview of major advances achieved by applying biotechnology to venom systems. This review therefore considers the methods, insights, and potential future developments of biotechnological applications in the field of venom research. We follow the levels of biological organization and structure, starting with the methods used to study the genomic blueprint and genetic machinery of venoms, followed gene products and their functional phenotypes. We argue that biotechnology can answer some of the most urgent questions in venom research, particularly when multiple approaches are combined together, and with other venomics technologies.

Glucosinolates, a natural chemical arsenal: More to tell than the myrosinase story.

Glucosinolates are a group of thioglucosides that belong to the class of plant nitrogen-containing natural products. So far, very little biological activity has been associated with intact glucosinolates. The hydrolysis of glucosinolates has, for long, attracted attention because of the potent biological activity of the hydrolysis products. From allelopathic to antiparasitic, antimicrobial and antineoplastic effects, the activity spectrum of the degradation products of typical glucosinolates has been the subject of much research. The present review seeks to address the various means of glucosinolate degradation (thermal, enzymatic, or chemical degradation) and the ensuing products. It also aims to draw a comparative profile of the various antimicrobial effects of these degradation products to provide a further understanding of the biological function of these important compounds.

Diverse chemical modifications of the chlorogenic acid composition of Viscum combreticola Engl.: A premise for the state of readiness against excessive sunlight exposure.

Mistletoe plants that are positioned on the canopy of their hosts are more susceptible to UV radiation exposure. These aerial plants are resistant to damage by UV radiation due the presence of epidermal constituents such as the cuticle, cork layer, trichomes and antioxidant secondary metabolites. In response to the photo-oxidative stress associated with UV exposure, plants generally deploy photo-protective responsive mechanisms that involve the biosynthesis of UV absorbing phenolic compounds such as chlorogenic acids (CGAs). The hydroxycinnamic acid moieties of these CGAs are predominantly in the trans configuration, naturally. However, excessive sunlight exposure of plants containing these compounds can result in geometrical isomerisation, characterized by the formation of cis isomers. Therefore, in this study, the effect of UV light radiation on the CGA composition of Viscum combreticola Engl. (Santalacaeae) plants using an in vitro model was unravelled through UHPLC-q-TOF-MS-based metabolic profiling. Interestingly, the findings of this study revealed that this plant has a diverse chemical composition of CGAs that is characterized by epimerization, monoacylation, homodiacylation and heterodiacylation of the quinic acid (QA), thereby, contributing to the state of readiness in these plants against sunlight or UV exposure. In addition to the commonly reported cinnamoyl containing heterodiacylated CGAs, hydroxybenzoyl containing heterodiacylated CGAs were also reported in this study. Moreover, cis isomers (24 in total) of some CGAs were identified in the non-irradiated samples and the formation of these compounds has been reported to help plants in the mitigation of photo-oxidative stress. An additional 28 cis isomers of CGAs and HCA derivatives were observed in the UV-irradiated samples, hence, further increasing the complexity of the metabolome of this plant, with a total of 108 compounds identified in this study. The presence of epimers, positional and geometrical isomers of these compounds could be a biochemical strategy to maximize the chemical arsenal of this plant to withstand the photo-oxidative stress posed by UV radiation from the sunlight. Due to purported pharmacological properties associated with the identified compounds these parasitic plants can be a rich source of prospective therapeutic compounds that can be employed as drug discovery leads. Moreover, UV radiation might be essential in future to produce potent drugs since plants naturally produce these compounds in low quantities.

Comparative analysis of assembly algorithms to optimize biosynthetic gene cluster identification in novel marine actinomycete genomes

Many marine sponges harbor dense communities of microbes that aid in the chemical defense of these nonmotile hosts. Metabolites that comprise this chemical arsenal can have pharmaceutically-relevant activities such as antibacterial, antiviral, antifungal and anticancer properties. Previous investigation of the Caribbean giant barrel sponge Xestospongia muta revealed a microbial community including novel Actinobacteria, a phylum well known for its production of antibiotic compounds. This novel assemblage was investigated for its ability to produce compounds that inhibit M. tuberculosis by using a bioinformatics approach. Microbial extracts were tested for their ability to inhibit growth of M. tb and genomes of the 11 strains that showed anti-M. tb activity including Micrococcus (n=2), Micromonospora (n=4), Streptomyces (n=3), and Brevibacterium spp. (n=2) were sequenced by using Illumina MiSeq. Three assembly algorithms/pipelines (SPAdes, A5-miseq and Shovill) were compared for their ability to construct contigs with minimal gaps to maximize the probability of identifying complete biosynthetic gene clusters (BGCs) present in the genomes. Although A5-miseq and Shovill usually assembled raw reads into the fewest contigs, after necessary post-assembly filtering, SPAdes generally produced the most complete genomes with the fewest contigs. This study revealed the strengths and weaknesses of the different assemblers based on their ease of use and ability to be manipulated based on output format. None of the assembly methods handle contamination well and high-quality DNA is a prerequisite. BGCs of compounds with known anti-TB activity were identified in all Micromonospora and Streptomyces strains (genomes &amp;gt; 5 Mb), while no such BGCs were identified in Micrococcus or Brevibacterium strains (genomes &amp;lt; 5 Mb). The majority of the putative BGCs identified were located on contig edges, emphasizing the inability of short-read assemblers to resolve repeat regions and supporting the need for long-read sequencing to fully resolve BGCs.

Cleavage Off-Loading and Post-assembly-Line Conversions Yield Products with Unusual Termini during Biosynthesis.

Piscibactins and photoxenobactins are metallophores and virulence factors, whose biosynthetic gene cluster, termed pxb, is the most prevalent polyketide synthase/non-ribosomal peptide synthetase hybrid cluster across entomopathogenic bacteria. They are structurally similar to yersiniabactin, which contributes to the virulence of the human pathogen Yersinia pestis. However, the pxb-derived products feature various chain lengths and unusual carboxamide, thiocarboxylic acid, and dithioperoxoate termini, which are rarely found in thiotemplated biosyntheses. Here, we characterize the pxb biosynthetic logic by gene deletions, site-directed mutagenesis, and isotope labeling experiments. Notably, we propose that it involves (1) heterocyclization domains with various catalytic efficiencies catalyzing thiazoline and amide/thioester bond formation and (2) putative C–N and C–S bond cleavage off-loading manners, which lead to products with different chain lengths and usual termini. Additionally, the post-assembly-line spontaneous conversions of the biosynthetic end product contribute to production titers of the other products in the culture medium. This study broadens our knowledge of thiotemplated biosynthesis and how bacterial host generate a chemical arsenal.

Evaluation of the High-Throughput Screening System for Assessing the Behavioral Response of FemaleAedes aegyptito Natural Products

As part of an arbovirus vector control strategy, chemical control continues to be a mainstay in mitigating the burden of disease. The current arsenal of chemicals used for this purpose, however, are becoming challenged rapidly because of issues of insecticide resistance and environmental pressure. Newer, environmentally friendly actives are of interest to supplement aging chemistries; therefore efforts to screen compounds for insecticidal activity are warranted. This study evaluated the efficacy of the high-throughput screening system (HITSS) for measuring the behavior-modifying actions of Brazilian Cerrado plant extracts, oils, and other compounds against Aedes aegypti. Different concentrations were evaluated, with 8 of 34 samples tested demonstrating either contact irritancy, spatial repellency, or attractiveness. We concluded several natural products screened in this study showed promise for use against mosquito vectors like Ae. aegypti, and that the compact modular HITSS assay constitutes a robust tool for measuring the behavioral responses of mosquitoes in the search for novel insecticides derived from natural products.

Molecular physiology of pumiliotoxin sequestration in a poison frog.

Poison frogs bioaccumulate alkaloids for chemical defense from their arthropod diet. Although many alkaloids are accumulated without modification, some poison frog species can metabolize pumiliotoxin (PTX 251D) into the more potent allopumiliotoxin (aPTX 267A). Despite extensive research characterizing the chemical arsenal of poison frogs, the physiological mechanisms involved in the sequestration and metabolism of individual alkaloids remain unclear. We first performed a feeding experiment with the Dyeing poison frog (Dendrobates tinctorius) to ask if this species can metabolize PTX 251D into aPTX 267A and what gene expression changes are associated with PTX 251D exposure in the intestines, liver, and skin. We found that D. tinctorius can metabolize PTX 251D into aPTX 267A, and that PTX 251D exposure changed the expression level of genes involved in immune system function and small molecule metabolism and transport. To better understand the functional significance of these changes in gene expression, we then conducted a series of high-throughput screens to determine the molecular targets of PTX 251D and identify potential proteins responsible for metabolism of PTX 251D into aPTX 267A. Although screens of PTX 251D binding human voltage-gated ion channels and G-protein coupled receptors were inconclusive, we identified human CYP2D6 as a rapid metabolizer of PTX 251D in a cytochrome P450 screen. Furthermore, a CYP2D6-like gene had increased expression in the intestines of animals fed PTX, suggesting this protein may be involved in PTX metabolism. These results show that individual alkaloids can modify gene expression across tissues, including genes involved in alkaloid metabolism. More broadly, this work suggests that specific alkaloid classes in wild diets may induce physiological changes for targeted accumulation and metabolism.

Tonoplast and Peroxisome Targeting of γ-tocopherol N-methyltransferase Homologs Involved in the Synthesis of Monoterpene Indole Alkaloids.

Many plant species from the Apocynaceae, Loganiaceae and Rubiaceae families evolved a specialized metabolism leading to the synthesis of a broad palette of monoterpene indole alkaloids (MIAs). These compounds are believed to constitute a cornerstone of the plant chemical arsenal but above all several MIAs display pharmacological properties that have been exploited for decades by humans to treat various diseases. It is established that MIAs are produced in planta due to complex biosynthetic pathways engaging a multitude of specialized enzymes but also a complex tissue and subcellular organization. In this context, N-methyltransferases (NMTs) represent an important family of enzymes indispensable for MIA biosynthesis but their characterization has always remained challenging. In particular, little is known about the subcellular localization of NMTs in MIA-producing plants. Here, we performed an extensive analysis on the subcellular localization of NMTs from four distinct medicinal plants but also experimentally validated that two putative NMTs from Catharanthus roseus exhibit NMT activity. Apart from providing unprecedented data regarding the targeting of these enzymes in planta, our results point out an additional layer of complexity to the subcellular organization of the MIA biosynthetic pathway by introducing tonoplast and peroxisome as new actors of the final steps of MIA biosynthesis.

Escherichia coli small molecule metabolism at the host-microorganism interface.

Escherichia coli are a common component of the human microbiota, and isolates exhibit probiotic, commensal and pathogenic roles in the host. E. coli members often use diverse small molecule chemistry to regulate intrabacterial, intermicrobial and host-bacterial interactions. While E. coli are considered to be a well-studied model organism in biology, much of their chemical arsenal has only more recently been defined, and much remains to be explored. Here we describe chemical signaling systems in E. coli in the context of the broader field of metabolism at the host-bacteria interface and the role of this signaling in disease modulation.

Effect of Spatial Variation on Defensive Substances of Constrictotermes Cyphergaster Soldiers (Blattaria, Isoptera).

The composition of chemical weaponry of termite soldiers show interspecific and intraspecific variation. However, spatial effects on the qualitative and quantitative compositions of these substances in Neotropical termites are poorly known. Hexane extracts of heads and the defensive secretion of soldiers of Constrictotermes cyphergaster from four localities in Northeast Brazil were analyzed by gas chromatography and mass spectrometry. Chemical analysis allowed the detection of 54 compounds from the head extract and from the direct extraction of the defensive secretion of soldiers, and the percentage of common substances and the presence of exclusive substances varied depending on the spatial distance between colonies. The profile of the chemical armament of soldiers consists basically of terpenoids: monoterpenes (45.53%-71.97 - for head extract and 57.41% - 78.56 for secretion) and sesquiterpenes (19.69% - 35.78% for head extract and 18.41% - 33.31%for secretion). In general, the main component of the chemical arsenal, regardless of the methodology used for extraction, was α-pinene (27.98-50.44%). Two chemotypes were identified based on chemical differences between populations of ecoregions with distinct spatial-environmental and climate characteristics: (1) α-pinene <33%; (2) α-pinene >33% for both extracts (head and secretion). The results reveal a similar pattern of chemical differentiation for soldiers in both extracts (head and secretion), with increasing differences as a function of distance between the analyzed colonies.

Chemical repertoire and biosynthetic machinery of the Aspergillus flavus secondary metabolome: A review.

Filamentous fungi represent a rich source of extrolites, including secondary metabolites (SMs) comprising a great variety of astonishing structures and interesting bioactivities. State-of-the-art techniques in genome mining, genetic manipulation, and secondary metabolomics have enabled the scientific community to better elucidate and more deeply appreciate the genetic and biosynthetic chemical arsenal of these microorganisms. Aspergillus flavus is best known as a contaminant of food and feed commodities and a producer of the carcinogenic family of SMs, aflatoxins. This fungus produces many SMs including polyketides, ribosomal and nonribosomal peptides, terpenoids, and other hybrid molecules. This review will discuss the chemical diversity, biosynthetic pathways, and biological/ecological role of A. flavus SMs, as well as their significance concerning food safety and security.

The Genetic Architecture of Plant Defense Trade-offs in a Common Monkeyflower.

Determining how adaptive combinations of traits arose requires understanding the prevalence and scope of genetic constraints. Frequently observed phenotypic correlations between plant growth, defenses, and/or reproductive timing have led researchers to suggest that pleiotropy or strong genetic linkage between variants affecting independent traits is pervasive. Alternatively, these correlations could arise via independent mutations in different genes for each trait and extensive correlational selection. Here we evaluate these alternatives by conducting a quantitative trait loci (QTL) mapping experiment involving a cross between 2 populations of common monkeyflower (Mimulus guttatus) that differ in growth rate as well as total concentration and arsenal composition of plant defense compounds, phenylpropanoid glycosides (PPGs). We find no evidence that pleiotropy underlies correlations between defense and growth rate. However, there is a strong genetic correlation between levels of total PPGs and flowering time that is largely attributable to a single shared QTL. While this result suggests a role for pleiotropy/close linkage, several other QTLs also contribute to variation in total PPGs. Additionally, divergent PPG arsenals are influenced by a number of smaller-effect QTLs that each underlie variation in 1 or 2 PPGs. This result indicates that chemical defense arsenals can be finely adapted to biotic environments despite sharing a common biochemical precursor. Together, our results show correlations between defense and life-history traits are influenced by pleiotropy or genetic linkage, but genetic constraints may have limited impact on future evolutionary responses, as a substantial proportion of variation in each trait is controlled by independent loci.

Structural venomics reveals evolution of a complex venom by duplication and diversification of an ancient peptide-encoding gene

Spiders are one of the most successful venomous animals, with more than 48,000 described species. Most spider venoms are dominated by cysteine-rich peptides with a diverse range of pharmacological activities. Some spider venoms contain thousands of unique peptides, but little is known about the mechanisms used to generate such complex chemical arsenals. We used an integrated transcriptomic, proteomic, and structural biology approach to demonstrate that the lethal Australian funnel-web spider produces 33 superfamilies of venom peptides and proteins. Twenty-six of the 33 superfamilies are disulfide-rich peptides, and we show that 15 of these are knottins that contribute >90% of the venom proteome. NMR analyses revealed that most of these disulfide-rich peptides are structurally related and range in complexity from simple to highly elaborated knottin domains, as well as double-knot toxins, that likely evolved from a single ancestral toxin gene.

HCN emission by a Polydesmid Millipede Detected Remotely by Reactive Adsorption on Gold Nanoparticles Followed by Laser Desorption/Ionization Mass Spectrometry (LDI-MS).

Hydrocyanic acid (HCN) is a well-known defensive allomone in the chemical arsenal of millipedes in the order Polydesmida. The presence of HCN in the headspace vapor of adult Xystocheir dissecta (Wood, 1867), a common millipede from the San Francisco Bay Area, was traced by laser desorption/ionization-mass spectrometry (LDI-MS). To accomplish this, the headspace vapor surrounding caged, live millipedes was allowed to diffuse passively over gold-nanoparticle (AuNP) deposits placed at various distances from the emitting source. The stainless steel plates with AuNP deposits were removed and irradiated by a 355-nm laser. The gaseous ions generated in this way were detected by time-of-flight mass spectrometry. The intensity of the mass spectrometric peak detected at m/z 249 for the Au(CN)2- complex anion was compared to that of the residual Au- signal (m/z 197). Using this procedure, HCN vapors produced by the live millipedes could be detected up to 50 cm away from the source. Furthermore, the addition of H2O2, as an internal oxygen source for the gold cyanidation reaction that takes place in the AuNP deposits, significantly increased the detection sensitivity. Using the modified H2O2 addition procedure, HCN could now be detected at 80 cm from the source. Moreover, we found a decreasing intensity ratio of the Au(CN)2-/Au- signals as the distance from the emitting source increased, following an exponential-decay distribution as predicted by Fick's law of diffusion. Graphical abstract.

Biosynthetic Origin of Complex Terpenoid Mixtures by Multiproduct Enzymes, Metal Cofactors, and Substrate Isomers

Terpenoids form a substantial portion of chemical diversity in nature. The enormous terpenoid diversity of more than 80,000 compounds is supported by the multisubstrate and multiproduct nature of certain enzymes from the various terpene synthases and terpene cyclases. These highly versatile enzymes are not only able to accept multiple substrates in their active site, but also simultaneously catalyze multiple reactions to the resultant multiple products. Interestingly, apart from the substrates and catalytic mechanisms, multiple regulation factors are able to alter the product profile of multiproduct terpene synthases. Simple variations in cellular conditions by changes in metal cofactors, assay pH, temperature and substrate geometry lead to significant shifts in product profiles. Switch in substrate stereochemistry for multiproduct terpene synthases in some case shows enhanced biocatalysis and in others initiates even a novel cyclization cascade. Hence, organisms can get access to a greater chemodiversity and avoid the expensive process of developing new biocatalysts just by simple changes in the cellular environment. This possibility of modulating chemical diversity provides immobile plants in the same generation access to an enhanced chemical arsenal for defense and communication by simply altering cofactors, pH level, and temperature and substrate geometry.

Metagenomic Exploration of the Marine Sponge Mycale hentscheli Uncovers Multiple Polyketide-Producing Bacterial Symbionts.

Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metagenomic investigation of Mycale hentscheli, a chemically gifted marine sponge that possesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short- and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemotypes. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holobiont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These results suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized.IMPORTANCEMycale hentscheli is a marine sponge that is rich in bioactive small molecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing microbe. We also find that the microbiome of M. hentscheli is stably maintained among individuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.